Nitric oxide synthase inhibitor phosphate salt

ABSTRACT

A compound of formula (I): namely (2S)-2-amino-4-{[2-(etha-nimidoylamino)ethyl]thio}butanoic acid, compound with prosphoric acid, or a solvate or physiologically functional derivative thereof, is useful as a relatively non-hygroscopic selective inhibitor of inducible nitric oxice synthase.

[0001] The present invention relates to novel amidino compounds, to aprocess for their manufacture, to pharmaceutical compositions containingthem, and to their use in therapy, in particular their use as selectiveinhibitors of inducible nitric oxide synthase.

[0002] Nitric oxide is the endogenous stimulator of the solubleguanylate cyclase enzyme and is involved in a number of biologicalactions. Excess nitric oxide production is also thought to be involvedin a number of conditions, including septic shock and many inflammatorydiseases. The biochemical synthesis of nitric oxide from L-arginine iscatalysed by the enzyme NO synthase. Many inhibitors of NO synthase havebeen described and proposed for therapeutic use.

[0003] More recently, it has been an object in this field to provide NOsynthase inhibitors displaying selectivity for either inducible NOsynthase (iNOS) or neuronal NO synthase (nNOS) over endothelial NOsynthase (eNOS).

[0004] Thus WO98/30537 describes selective NO synthase inhibitors offormula

[0005] or a salt, solvate, or physiologically functional derivativethereof.

[0006] Suitable salts according to WO98/30537 include those formed withboth organic and inorganic acids or bases. Pharmaceutically acceptableacid addition salts include those formed from hydrochloric, hydrobromic,sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic,trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic,methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic,and isethionic acids. Pharmaceutically acceptable base salts includeammonium salts, alkali metal salts such as those of sodium andpotassium, alkaline earth metal salts such as those of calcium andmagnesium and salts with organic bases such as dicyclohexyl amine andN-methyl-D-glucamine. Hydrochloride salts are exemplified and are statedto be hygroscopic.

[0007] The present invention seeks to provide a selective iNOS inhibitorwhich is considerably less hygroscopic than, for example, thehydrochloride salts exemplified in WO98/30537.

[0008] We have now found compounds falling within the scope ofWO98/30537 which are selective iNOS inhibitors and display advantages,including being relatively non-hygroscopic. A compound which isrelatively non-hygroscopic is useful because it does not readily absorbmoisture from the atmosphere and is therefore easier to isolate,formulate and handle.

[0009] According to the present invention there is provided a compoundof formula (I)

[0010] or a solvate or physiologically functional derivative thereof.

[0011] The present invention therefore provides(2S)-2-amino-4-{[2-(ethanimidoylamino)ethyl]thio}butanoic acid, compoundwith phosphoric acid, or a solvate (preferably hydrates) orphysiologically functional derivative thereof. The present inventionincludes all polymorphs of this compound. Preferably each molecule ofthe compound of formula (I) is associated with at least one molecule ofwater, such as 1, 2 or 3 water molecules, especially 1 or 3 watermolecules. In a particularly preferred aspect, the present inventionprovides (2S)-2-amino-4-{[2-(ethanimidoylamino)ethyl]thio}butanoic acid,compound with phosphoric acid, (1:1) hydrate.

[0012] It will be appreciated by those skilled in the art thatphosphoric acid exists in more than one form. The preferred form for usein the context of the present invention is orthophosphoric acid.

[0013] Preferably, the compound of formula (I) does not substantiallydeliquesce (ie rapidly take up atmospheric moisture) below a relativehumidity of 60% at 25 degrees C. More preferably, the compound offormula (I) does not substantially deliquesce below a relative humidityof 70% at 25 degrees C.

[0014] Preferably, the compound of formula (I) does not undergo a masschange via adsorption of more than 5% w/w at a relative humidity of 70%at 25 degrees C. More preferably, the compound of formula (I) does notundergo a mass change via adsorption of more than 2% w/w at a relativehumidity of 70% at 25 degrees C. Most preferably, the compound offormula (I) does not undergo a mass change via adsorption of more than1% w/w at a relative humidity of 70% at 25 degrees C.

[0015] Solvates of the compound of formula (I) which are suitable foruse in medicine are those wherein the associated solvent ispharmaceutically acceptable. However, solvates havingnon-pharmaceutically acceptable associated solvents are within the scopeof the present invention, for example, for use as intermediates in thepreparation of the compound of formula (I) and its solvates, andphysiologically functional derivatives.

[0016] By the term “physiologically functional derivative” is meant achemical derivative of the compound of formula (I) having the samephysiological function as the compound of formula (I), for example, bybeing convertible in the body thereto. According to the presentinvention, examples of physiologically functional derivatives includeesters, amides, and carbamates; preferably esters and amides.

[0017] Pharmaceutically acceptable esters and amides of the compound offormula (I) may have the acid group converted to a C₁₋₆ alkyl, aryl,aryl C₁₋₆ alkyl, or amino acid ester or amide. Pharmaceuticallyacceptable amides and carbamates of the compound of formula (I) may havean amino group converted to a C₁₋₆ alkyl, aryl, aryl C₁₋₆ alkyl, oramino acid amide or carbamate.

[0018] As mentioned above, the compound of formula (I) is an inhibitorof NO synthase. WO98/30537 demonstrates this generally for compounds ofthe following formula

[0019] or a salt, solvate, or physiologically functional derivativethereof: phosphate salts are not exemplified.

[0020] The compound of formula (I) and its pharmaceutically acceptablesolvates and physiologically functional derivatives have use in theprophylaxis and treatment of clinical conditions for which an inhibitorof NO synthase is indicated, in particular an inhibitor of iNOS. Suchconditions include inflammatory conditions, shock states, immunedisorders, and disorders of gastrointestinal motility. The compound offormula (I) and its pharmaceutically acceptable solvates, andphysiologically functional derivatives thereof may also be of use in theprophylaxis and treatment of diseases of the central nervous systemincluding migraine and metabolic disorders including dyslipidemia.

[0021] By shock states is meant those resulting from overproduction ofNO, such as septic shock, haemorrhagic shock, traumatic shock, or shockcaused by fulminant hepatic failure or by therapy with cytokines such asTNF, IL-1 and IL-2 or therapy with cytokine-inducing agents, for example5,6-dimethylxanthenone acetic acid.

[0022] Examples of inflammatory conditions and immune disorders includethose of the joint, particularly arthritis (e.g. rheumatoid arthritis,osteoarthritis, prosthetic joint failure), or the gastrointestinal tract(e.g. ulcerative colitis, Crohn's disease, and other inflammatory boweldiseases, gastritis and mucosal inflammation resulting from infection,the enteropathy provoked by non-steroidal anti-inflammatory drugs), ofthe airways (e.g. adult respiratory distress syndrome, asthma, cysticfibrosis, upper respiratory tract inflammatory disease (e.g. rhinitissuch as allergic rhinitis) or chronic obstructive pulmonary disease), ofthe heart (e.g. myocarditis), of nervous tissue (e.g. multiplesclerosis), of the pancreas (e.g. diabetes melitus and complicationsthereof), of the kidney (e.g. glomerulonephritis), of the skin (e.g.dermatitis, psoriasis, eczema, urticaria), of the eye (e.g. glaucoma) aswell as of transplanted organs (e.g. rejection) and multi-organ diseases(e.g. systemic lupus erythematosis) and inflammatory sequelae of viralor bacterial infections.

[0023] Furthermore, there is evidence for overproduction of NO by iNOSin atherosclerosis and following hypoxic or ischaemic insults (with orwithout reperfusion), for example in the brain or in ischaemic heartdisease.

[0024] Disorders of gastrointestinal motility include ileus, for examplepost-operative ileus and ileus during sepsis.

[0025] By diseases of the central nervous system is meant those forwhich overproduction of NO is implicated, for example migraine,psychosis, anxiety, schizophrenia, sleep disorders, cerebral ischaemia,CNS trauma, epilepsy, multiple sclerosis, AIDS dementia, chronicneurodegenerative disease (e.g. Lewy Body Dementia, Huntington'sdisease, Parkinson's disease, or Alzheimer's disease) and acute andchronic pain, and conditions in which non-adrenergic non-cholinergicnerve may be implicated such as priapism, obesity and hyperphagia.

[0026] Examples of acute pain include musculoskeletal pain, postoperative pain and surgical pain. Examples of chronic pain includechronic inflammatory pain (e.g. rheumatoid arthritis andosteoarthritis), neuropathic pain (e.g. post herpetic neuralgia,diabetic neuropathies associated with diabetes, trigeminal neuralgia,pain associated with functional bowel disorders, e.g. irritable bowelsyndrome, non cardiac chest pain and sympathetically maintained pain)and pain associated with cancer and fibromyalgia.

[0027] Overproduction of NO is implicated in metabolic disorders throughits effect on lipoprotein lipase activity causing hypertriglyceridemia.Selective inhibitors of iNOS will be useful in metabolic conditionswhere NO over-production is implicated, such as dyslipidaemia.

[0028] Furthermore, inhibition of NO synthase may be of advantage inpreventing the lymphocyte loss associated with HIV infection, inincreasing the radiosensitivity of tumours during radiotherapy and inreducing tumour growth, tumour progression, angiogenesis, andmetastasis.

[0029] Accordingly, the present invention provides a method for theprophylaxis or treatment of a clinical condition in a mammal, such as ahuman, for which an inhibitor of nitric oxide synthase, for example, aniNOS inhibitor is indicated, which comprises administration of atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable solvate or physiologically functionalderivative thereof. In particular, the present invention provides amethod for the prophylaxis or treatment of an inflammatory and/or immunedisorder, such as arthritis, allergic rhinitis or asthma. In a furtherpreferred aspect the present invention provides a method for theprophylaxis or treatment of a clinical condition selected from pain,migraine, ileus and irritable bowel syndrome.

[0030] In the alternative, there is also provided a compound of formula(I) or a pharmaceutically acceptable solvate or physiologicallyfunctional derivative thereof for use in medical therapy, particularlyfor use in the prophylaxis or treatment of a clinical condition in amammal, such as a human, for which an inhibitor of nitric oxidesynthase, for example an iNOS inhibitor, is indicated. In particular,there is provided a compound of formula (I) or a pharmaceuticallyacceptable solvate or physiologically functional derivative thereof forthe prophylaxis or treatment of an inflammatory and/or immune disorder,such as arthritis, allergic rhinitis or asthma. In a further preferredaspect, there is provided a compound of formula (I) or apharmaceutically acceptable solvate or physiologically functionalderivative thereof for the prophylaxis or treatment of pain, migraine,ileus or irritable bowel syndrome.

[0031] The amount of the compound of formula (I), or a pharmaceuticallyacceptable solvate or physiologically functional derivative thereofwhich is required to achieve a therapeutic effect will, of course, varywith the particular compound, the route of administration, the subjectunder treatment, and the particular disorder or disease being treated.The compounds of the invention may be administered orally or viainjection at a dose of from 0.001 to 200 mg/kg per day, preferably 0.01to 20 mg/kg per day. The dose range for adult humans is generally from0.1 mg to 10 g/day and preferably 1 mg to 1 g/day. Tablets or otherforms of presentation provided in discrete units may convenientlycontain an amount of compound of the invention which is effective atsuch dosage or as a multiple of the same, for instance, units containing0.1 mg to 500 mg, usually around 1 mg to 200 mg.

[0032] While it is possible for the compound of formula (I), or apharmaceutically acceptable solvate or physiologically functionalderivative thereof to be administered alone, it is preferable to presentthem as a pharmaceutical formulation.

[0033] Accordingly, the present invention further provides apharmaceutical formulation comprising a compound of formula (I) or apharmaceutically acceptable solvate or physiologically functionalderivative thereof, and a pharmaceutically acceptable carrier orexcipient, and optionally one or more other therapeutic ingredients.

[0034] The present invention also provides the use of a compound offormula (I), or a pharmaceutically acceptable solvate or physiologicallyfunctional derivative thereof in the manufacture of a medicament for theprophylaxis or treatment of a clinical condition for which an inhibitorof nitric oxide synthase, for example an iNOS inhibitor, is indicated,for example an inflammatory and/or immune disorder, such as arthritis,allergic rhinitis or asthma. In a further preferred aspect, there isprovided a compound of formula (I), or a pharmaceutically acceptablesolvate or physiologically functional derivative thereof in themanufacture of a medicament for the prophylaxis or treatment of aclinical condition selected from pain, migraine, ileus and irritablebowel syndrome.

[0035] Hereinafter, the term “active ingredient” means a compound offormula (I), or a pharmaceutically acceptable solvate or physiologicallyfunctional derivative thereof.

[0036] The formulations include those suitable for oral, parenteral(including subcutaneous, intradermal, intramuscular, intravenous andintraarticular), inhalation (including fine particle dusts or mistswhich may be generated by means of various types of metered dosepressurised aerosols, nebulisers or insufflators), rectal and topical(including dermal, buccal, sublingual and intraocular) administrationalthough the most suitable route may depend upon for example thecondition and disorder of the recipient. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing the active ingredient into association with thecarrier which constitutes one or more accessory ingredients. In generalthe formulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

[0037] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

[0038] A tablet may be made by compression or moulding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

[0039] Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example saline or water-for-injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

[0040] Formulations for rectal administration may be presented as asuppository with the usual carriers such as cocoa butter or polyethyleneglycol.

[0041] Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavoured basis such as sucrose and acacia ortragacanth, and pastilles comprising the active ingredient in a basissuch as gelatin and glycerin or sucrose and acacia.

[0042] The relatively non-hygroscopic nature of the compound of formula(I) renders it particularly suitable for administration in solid forme.g. as tablets.

[0043] In a preferred aspect the present invention provides a soliddosage form comprising the compound of formula (I) or a solvate orphysiologically functional derivative thereof.

[0044] Preferred unit dosage formulations are those containing aneffective dose, as hereinbefore recited, or an appropriate fractionthereof, of the active ingredient.

[0045] It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of this invention mayinclude other agents conventional in the art having regard to the typeof formulation in question, for example those suitable for oraladministration may include flavouring agents.

[0046] According to a further aspect of the invention, there is provideda process for preparing a compound of formula (I) or a solvate orphysiologically functional derivative thereof which comprises:

[0047] (i) reacting a compound of formula (II)

[0048] or an enantiomer, a salt, or a protected derivative thereof, witha compound of formula (III)

[0049] or a salt thereof, wherein L is a leaving group, most suitably aC₁₋₆ alkoxy group (for example ethoxy), an alkylthio group, anaralkylthio group, an arylthio group (for example benzylthio), a 1- or2-naphthylmethylthio group or a heterocycle group; followed by thefollowing steps in any order:

[0050] (ii) converting the resulting compound to the monophosphate salt;

[0051] (iii) optional removal of any protecting groups;

[0052] (iv) optional separation of an enantiomer from a mixture ofenantiomers;

[0053] (v) optional conversion of the product to a corresponding solvateor physiologically functional derivative thereof.

[0054] Preferably compounds of formula (I) prepared by this method arepurified by recrystallization from a suitable solvent, such as anaqueous solvent, for example, water, an aqueous alcohol or aqueousacetonitrile, or a polar organic solvent, for exampleN,N-dimethylformamide, dimethylsulphoxide, dimethylacetamide or1-methyl-2-pyrrolidinone. A preferred solvent for recrystallization isaqueous ethanol.

[0055] In a preferred embodiment, the compound that results from thereaction of the compounds of formulae (II) and (III) is reacted withphosphoric acid, preferably orthophosphoric acid, to form the compoundof formula (I).

[0056] Preferably, the step of converting the resulting compound to thephosphate salt comprises a biphasic reaction using toluene and aqueousphosphoric acid.

[0057] In a preferred embodiment, following step (i): toluene is addedto an aqueous layer resulting from the reaction of the compound offormula (II) or an enantiomer, a salt, or a protected derivative thereofwith the compound of formula (III) or a salt thereof to form a biphasicmixture; a toluene layer is separated from this mixture; an aqueoussolution of phosphoric acid is added to the toluene layer to form afurther biphasic mixture; and an aqueous layer is separated from thisfurther mixture.

[0058] When L is C₁₋₆ alkoxy, the reaction in step (i) above may beeffected in solution at alkaline pH, for example pH 8 to 11, suitably atpH p.5 to 10.5, and at a low temperature, for example −5° C. to 25° C.When L is an alkylthio, aralkylthio, or arylthio group, the reaction maybe effected in an organic solvent e.g. dimethylformamide,tetrahydrofuran, ethyl acetate or a C₁₋₄ alcohol such as ethanol, at amoderate temperature e.g. 10 to 40° C., suitably at ambient temperature.

[0059] Preferably, the compound of formula (II) is

[0060] Preferably, the compound of formula (III) is

[0061] The compounds of formula (II) and derivatives thereof may beprepared by reacting a compound of formula (IV)

[0062] wherein X is a leaving group, most suitably a halo group such asBr, or an enatiomer, salt or protected derivative thereof, with thecompound of formula (V).

[0063] or a salt or protected derivative thereof.

[0064] Compounds of formula (III) and salts thereof may be prepared bymethods of organic chemistry well known to the person skilled in theart, for example, as described by Shearer et al in Tetrahedron Letters,1997, 38, 179-182.

[0065] The protecting groups used in the preparation of compounds offormula (I) may be used in a conventional manner, for example, usingmethods described in “Protective Groups in Organic Synthesis” byTheodora W Green, 2nd edition (John Wiley and Sons, 1991) which alsodescribes methods for the removal of such groups.

[0066] In the above reactions, primary amines are suitably protected ascarbamates, such as t-butoxycarbonyl or benzyloxycarbonyl groups whichmay be removed under acidic conditions, for example, by treatment withhydrochloric acid or hydrobromic acid, or by hydrogenolysis.

[0067] As will be appreciated by the person skilled in the art use ofsuch protecting groups may include orthogonal protection of amino groupsin the compounds of formula (II) to facilitate the selective removal ofone group in the presence of another, thus enabling selectivefunctionalisation of a single amino function. For example, abenzyloxycarbonyl group may be selectively removed by hydrogenolysis. Aperson skilled in the art will also appreciate other orthogonalprotection strategies, available by conventional means as described inTheodora W Green (vide supra).

[0068] The enantiomeric compounds of the invention may be obtained (a)by separation of the components of the corresponding racemic mixture,for example, by means of a chiral chromatography column, enzymaticresolution methods or preparing and separating suitablediastereoisomers, or (b) by direct synthesis from the appropriate chiralstarting materials by the methods described above.

[0069] Optional conversion of a compound of formula (I) to acorresponding solvate or physiologically functional derivative may beeffected by methods known to those skilled in the art. For example, thetrihydrate of the compound of formula (I) may be prepared from thecorresponding monohydrate via exposure to a high humidity environmentfor 24 hours.

[0070] The present invention will now be described by way of exampleonly with reference to the accompanying diagrammatic drawings in which:

[0071]FIG. 1 is a full isotherm plot at 25 degrees C of % targetrelative humidity against % weight change (w/w) showing moisturesorption and desorption for a compound of formula (I), monohydrate;

[0072]FIG. 2 is another full isotherm plot at 25 degrees C of % targetrelative humidity against % weight change (w/w) showing moisturesorption and desorption for the compound of formula (I), monohydrate;

[0073]FIG. 3 is a further full isotherm plot at 25 degrees C of % targetrelative humidity against % weight change (w/w) showing moisturesorption and desorption for the compound of formula (I), monohydrate;

[0074]FIG. 4 is a comparative moisture sorption isotherm plot at 25degrees C of % target relative humidity against % weight change for(2S)-2-amino4-{[2-(ethanimidoylamino)ethyl]thio}butanoic acid, compoundwith hydrochloric acid; and

[0075]FIG. 5 is full isotherm plot a 25° C. of % target relativehumidity against % weight change (w/w) showing moisture sorption anddesorption for the compound of formula (I), trihydrate. The sorption anddesorption traces are overlaid. The sorption isotherm started starts ata relative humidity of 30% increasing to 90%, followed by desorptionfrom 90% RH to 0% RH.

[0076]FIG. 6 is an X-ray diffraction pattern for a compound of formula(I) monohydrate.

[0077]FIG. 7 is an X-ray diffraction pattern for a compound of formula(I) trihydrate

EXAMPLE 1 Preparation of Compound of Formula (II)

[0078] A) Stage 1: Preparation of L-Homoserine Lactone

[0079] The reaction was carried out under a nitrogen atmosphere. Astirred suspension of L-methionine (1 mol eq., 1.0 wt) at roomtemperature in water (4 Vol), isopropyl alcohol (4 vol) and glacialacetic acid (1.6 vol) was prepared, to which was added bromoacetic acid(1.0 wt). The suspension was heated to 50° C. and held at thattemperature for 2 hours. The temperature was raised to ca. 82-85° C. andthe solution heated for a further 5 hours. The orange-brown solution wascooled and the solvents removed under reduced pressure. The thick, darkbrown slurry was heated (water bath temperature 90° C.) for two hourswhilst under vacuum. The slurry was allowed to cool then suspended in 4MHCl in dioxane (2 Vol) and stirred at room temperature for a minimumperiod of 3 hours. The crystalline solids were collected by filtrationand suspended in isopropyl alcohol (1.5 vol): the suspension was stirredfor at least 1 hour. The solids were collected again by filtration anddried in vacuo at 50-60° C. to give L-homoserine lactone as an off-whitecrystalline solid.

[0080] B) Stage 2: Preparation of (2S)-2-amino-4-bromobutanoic AcidHydrobromide

[0081] The reaction was carried out under a nitrogen atmosphere.L-Homoserine lactone (1.0 mol eq., 1.0 wt) was suspended and stirred inglacial acetic acid (2 Vol) at 20±5° C. under a static atmosphere of N₂.To the suspension was added (8 Vol) of a 45% w/v solution of hydrogenbromide in acetic acid. The contents were ramp heated to 52±1° C. over 4hours, and then stirred at 52±1° C. for 16 hours to give a white solidsuspended in a yellow or orange solution. The contents were cooled to20±2° C. and held at 20±2° C. for at least two hours to allow theproduct to fully precipitate. The solid was collected by filtration. Thecake was then washed, firstly with acetic acid (3×2 Vol) and thentert-butylmethyl ether (TBME; 3×2 vol). The product was dried toconstant weight in a vacuum oven at 45±5° C. and collected as awhite/off-white crystalline solid.

[0082] C) Stage 3: Preparation of tert-butyl(2S)-2-amino-4-bromobutanoate sulfate

[0083] The reaction was carried out under a nitrogen atmosphere.(2S)-2-amino-4-bromobutanoic acid hydrobromide (1 mol eq., 1.0 wt) wassuspended and stirred in tert-butyl acetate (10 vol.) at 5±3° C. for ca.1 hour under an atmosphere of N₂. To the suspension was added, dropwiseover ca. 30 minutes, concentrated sulphuric acid (1.2 eq, 0.445 wt),maintaining a contents temperature of 5±3° C. The contents were warmedto 18±3° C. and stirred at that temperature for 10 mins. Glacial aceticacid (2.5 vol) was added over ca. 10 minutes and then the reactionmixture was stirred vigorously until a clear solution was obtained (upto 3 hours). Stirring continued at 18±3° C. for at least 2 hours.Following the onset of crystallisation, the reaction mixture was agedfor at least 4 hours at 18±30° C. Toluene (5.5 vol) was then added over50 min. to the slurry. The resulting white mixture was stirred for 18hours at 18±3° C. before collecting the product by filtration. Thefilter cake was washed with ethyl acetate (2.5 vol). The damp productwas slurried with fresh ethyl acetate (10 vol) at 20±3° C. for 1 hour.The product was again collected by filtration and the filter cake waswashed with ethyl acetate (2×2.5 vol). The product was dried in a vacuumoven at 20±5° C.

[0084] D) Stage 4: Preparation of tert-butyl(2S)-4-bromo-2-[(tert-butoxycarbonyl)amino]butanoate

[0085] The reaction was carried out under a nitrogen atmosphere.Anhydrous sodium hydrogen carbonate (2.0 mol eq.) was vigorously stirredin water (3.0 vol) at 20±3° C. under a static N₂ atmosphere. Ethylacetate (3.0 vol) was added and the mixture was cooled to 5±2° C. Asolution of tert-butyl (2S)-2-amino-4-bromobutanoate sulfate (1.0 moleq., 1.0 wt) in water (2.0 vol) was added over ca. 20 minutes,maintaining a contents temperature of 5±2° C. A solution ofdi-t-butyidicarbonate (0.96 eq) in ethyl acetate (2.0 vol) was thenadded over 5 minutes to the reaction mixture, maintaining the contentsat 5±2° C. The biphasic mixture was allowed to warm to 20±3° C. and thenstirred for a further 3-5 hours. The layers were allowed to separateovernight. The aqueous layer was removed and the organic layer waswashed with brine (1×5 vol). The layers were separated and the organiclayer was dried over anhydrous MgSO₄, filtered and then concentratedunder reduced pressure to yield a colourless oil. The colourless oilsolidified on standing to a hard white solid.

[0086] E) Stage 5: Preparation of tert-butylS-(2-aminoethyl)-N-(tert-butoxycarbonyl)-L-homocysteinate.

[0087] The reaction was carried out under a nitrogen atmosphere.Anhydrous potassium carbonate (3.0 mol eq.) was added to a stirredsuspension of cysteamine hydrochloride (1.5 mol eq.) in methanol (5 vol)at 20±3° C., under an atmosphere of N₂. The mixture was stirred for 30minutes before a solution of tert-butyl(2S)-4-bromo-2-[(tert-butoxycarbonyl)amino]butanoate (1.0 mol eq., 1.0wt) in methanol (5 vol) was added dropwise maintaining the contentstemperature at 20±3° C. The reaction mixture was left stirring at 25±2°C. for 4 hours. The reaction mixture was concentrated under reducedpressure using a water bath at <30° C. to yield a white residue. Theresidue was partitioned between tert-butylmethyl ether (TBME; 8 vol) andwater (8 vol). After 10 min. thorough shaking/mixing, the layers wereallowed to separate. The aqueous phase was removed and the organic phasewas concentrated under reduced pressure using a water bath at <30° C. toapproximately half the volume (ca. 4 vol). Formic acid (1.0 mol eq.) wasadded, dropwise, to the stirred TBME solution maintaining a contentstemperature of 20±3° C. The reaction mixture was stirred at 20±3° C. forone hour during which time a white precipitate formed. The mixture wascooled to 5±2° C. for at least 16 hours. The product was collected byfiltration and then washed with additional TBME (2×1 vol). The solid wasdried to constant weight in a vacuum oven at 40±2° C. affording theproduct as a white crystalline solid.

EXAMPLE 2 Alternative Preparation of Compound of Formula (II)

[0088] (2S)-2-Amino-4-bromobutanoic acid hydrobromide (1.0 wt, 1.0 mol)was suspended and stirred in tert-butyl acetate (TBA, 5 vols) at ca. 5°C. under a static nitrogen atmosphere. Concentrated sulphuric acid (98%purity; 1.2 mol eq.) was added to the suspension over 20 min,maintaining the contents temperature ˜15° C. The contents were thenwarmed to around 20° C. and allowed to stir for at least 7 h at 20° C.After 1-2 h from the end of sulphuric acid addition, crystallisation wasobserved. 2M aqueous sodium hydroxide solution (8 mol eqs) was addedover ca. 60 min maintaining the contents temperature ca. 20° C. The pHof the aqueous was checked and found to be above 9. The layers wereseparated and the aqueous layer discarded. In a separate container,di-t-butyldicarbonate (99%; 0.95 mol eq.) was dissolved in TBME (2vols). This solution was added, over 20 min, to the TBA solution,maintaining the contents temperature below 20° C. The solution wasstirred at ca. 20° C. for 4 h.

[0089] 32% w/w NaOH (4 vols) was charged to the reactor and cooled toca. 10° C. Cysteamine hydrochloride (1.2 mol eq.) was added, maintainingthe contents temperature at 10-20° C. This mixture was then stirred forca. 30 min to allow for full neutralisation of the hydrochloride. TheAliquat 175 (methyl tributylammonium chloride; 75% w/w aqueous solution;0.05 mol) was added maintaining the contents temperature at 10-20° C.The TBA/TBME solution was added to the aqueous over 20 min maintainingthe temperature at ca. 20° C. The biphasic mixture was then stirred atca. 20° C. for ca. 20 h. Water (4 vols) was then added over ca. 10 minmaintaining the contents temperature at ca. 20° C. Stirring for 15 mingave 2 clear layers. The aqueous layer was separated and discarded. Theorganic layer is washed with water (2×4 vols). The organic solution wasdiluted by the addition of TBME (6-8 volumes). Formic acid (98% w/w; 0.8mol eq) was then added, over 20 min, to the stirred TBA/TBME solutionmaintaining a contents temperature of ca. 20° C. The reaction mixturewas cooled to −5° C. for 24 hours. The product was collected byfiltration and then washed with additional TBME (2×1 vol). The solid wasthen dried in a vacuum oven at 40±5° C. affording the product as a whitecrystalline solid. The reaction scheme of this method is shown below:

EXAMPLE 3 Preparation of Compound of Formula (III)

[0090]

[0091] The reaction was carried out under a nitrogen atmosphere.Ethanethioamide (1 wt, 1 eq.) was added to acetonitrile (14 vol) withstirring under a nitrogen atmosphere. The resultant slurry was heated to65° C. ±2° C. whereupon a yellow solution was formed. To this solutionwas added, dropwise, a separate solution of 1-(chloromethyl)naphthalene(2.47 wts, 1.05 eq.) in acetonitrile (3 vol), controlled in such amanner as to maintain the contents temperature at 60-650° C. Once theaddition was complete, a further portion of acetonitrile (1 vol) wasused as a line wash. The resultant solution was heated to 70° C. ±2° C.:crystallisation was observed within 10-20 minutes. The slurry was heatedat 70° C. ±9° C. for a total of 3 hours and then cooled to roomtemperature. The solid was filtered, and the cake was washed withacetonitrile (2×5 vol). The resulting white solid was dried in vacuo at45° C. ±50C for at least 16 hours.

EXAMPLE 4 Preparation of Compound of Formula (I) (Monohydrate)

[0092] The reaction was carried out under a nitrogen atmosphere. Amixture of tert-butylS-(2-aminoethyl)-N-(tert-butoxycarbonyl)-L-homocysteinate compound withformic acid (1:1) (a compound of formula II) (1.0 mol eq, 1.0 wt) and1-naphthylmethyl ethanimidothioate hydrochloride (a compound of formulaIII) (1.2 mol eq) was stirred in ethyl acetate (6 vol) at roomtemperature for 2 hours. Water (8.5 vol) was added and the mixture wasvigorously stirred for 30 mins. The layers were allowed to settle andthe aqueous layer containing tert-butylN-(tert-butoxycarbonyl)-S-[2-(ethanimidoylamino)ethyl]-L-homocysteinatehydrochloride was collected. The aqueous layer was cooled to 0° C. andtoluene (5 vol) was added. 32% w/w aqueous sodium hydroxide (2.5 mol eq)was slowly added, maintaining the temperature at 2° C. and the biphasicmixture was vigorously stirred for 10 min. The layers were separated,the aqueous layer was recharged to the vessel, and the toluene layerstored. Toluene (2.5 vol) was added to the aqueous layer and the mixturecooled to 0° C. 32% w/w aqueous sodium hydroxide (1.0 mol eq) was slowlyadded, maintaining the temperature at 0° C. and the biphasic mixture wasvigorously stirred for 5 minutes. The layers were separated, the aqueouslayer was discarded and the toluene layers containing tert-butylN-(tert-butoxycarbonyl)-S-[2-(ethanimidoylamino)ethyl]-L-homocysteinatewere combined in the vessel. An aqueous solution (2.5 vol) oforthophosphoric acid (1.5 mol eq) was added. The biphasic mixture wasvigorously stirred at 70° C. for 12 hours. Water (4 vol) was then addedand the mixture cooled to 40° C. The aqueous layer was separated and thepH was then adjusted to 5.5 using concentrated aqueous ammonia solutionmaintaining the temperature at 35° C. The resulting aqueous solution washeated to 75° C. and then ethanol (6.5 vol) added over 20 min.maintaining the temperature at 75° C. The solution was ramp cooled from75° C. to 53° C. over 30 minutes and seeded. It was held at 53° C. for 2hours and ramp cooled to 5° C. over 4 hours. The resulting slurry washeld at that temperature for 20 hours. The product was collected byfiltration and was washed with a 1:1 mixture of ethanol/water (2 vol)and then with ethanol (2 vol). A compound of formula (I), namely(2S)-2-amino-4-{[2-(ethanimidoylamino)ethyl]thio}butanoic acid, compoundwith phosphoric acid, monohydrate was dried at 60° C. in a vacuum toconstant weight.

[0093] The reaction scheme of this method is shown below.

[0094] The hygroscopicities of three samples of the compound of formula(I) monohydrate prepared in accordance with the above method were testedby measuring their % weight change (w/w) over a target relative humidityband of 0 to 90% at 25 degrees C. The results are shown in FIGS. 1 to 3.

[0095] To make these measurements, the following instruments andparameters were used:

[0096] Hiden IGASORP, Serial: IGA SA-040;

[0097] Hiden Intelligent Analyser, Model: HAS-022-120E, Serial:HALIGA-0042;

[0098] Flow=495 ml/min Isothermal Parameters: Analysis Mode: F1 WaitUntil:  97% Min Time:  10 minutes Max Time: 240 minutes M-Level: 0.2%Isothermal sequence: Temperature: 25° C. Adsorption/% Desorption/%  0 8010 70 20 60 30 50 40 40 50 30 60 20 70 10 80  0 90

[0099] It can be seen from FIGS. 1 to 3 that there is no significantuptake of atmospheric moisture below a relative humidity of 70%.

[0100] X-ray diffraction data for(2S)-2-amino-4-{[2-(ethanimidoylamino)ethyl]thio}butanoic acid, compoundwith orthophosphoric acid, (1:1) hydrate is shown in FIG. 6. Table 1below sets out the instrument and parameters used. Table 2 below setsout the peak listings. TABLE 1 Manufacturer Philips Analytical X-Ray B.V. The Netherlands Diffractometer Type PW1800 (PC-APD) Serial DY701 TubeAnode Cu LabdaAlpha1 1.54056 LabdaAlpha2 1.54439 RatioAlpha21 0.50000Divergence Slit Automatic Receiving Slit Fine Monochromator Used YESGenerator Voltage 40 Tube Current 45 File Date & Time 4-Oct-2000 3:23Data Angle Range (°2Θ) 6.0000-45.0000 Scan Step Size (°2Θ) 0.020 ScanType STEP Scan Step Time 4.00

[0101] TABLE 2 Angle (°2Θ) Relative Intensity (%) 7.2900 100 7.5100 19.48.5000 1 12.0650 0 14.4950 60 16.9250 2 18.4300 16 18.6950 2 20.3350 220.7400 1 21.1200 1 21.8250 6 22.2150 10 22.4500 12 23.4950 2 23.7100 424.1450 38 25.3450 2 25.8800 2 26.4950 3 26.6750 2 27.4650 7 27.9600 628.4050 2 29.0200 7 29.4950 1 30.1800 3 30.7400 1 31.8000 1 32.1800 232.5200 5 33.8400 1 34.4650 5 35.5200 1 35.8150 1 36.5950 5 37.8300 138.5550 1 39.2200 1 39.7850 4 40.9400 1 41.5450 1 42.0900 1 42.5700 143.7200 1 44.4700 2

EXAMPLE 5 Preparation of Compound of Formula (I) (Trihydrate)

[0102] Compound of formula (I) monohydrate prepared according to themethod of Example 4 (5 g) was exposed to a relative humidity of greaterthan 80% for 2 weeks. The product was analysed by X-ray powderdiffraction and shown to be the trihydrate. (FIG. 7)

[0103] X-ray diffraction data for(2S)-2-amino-4-{[2-(ethanimidoylamino)ethyl]thio}butanoic acid, compoundwith orthophosphoric acid, (1:3) hydrate is shown in FIG. 7. Table 3below sets out the instrument and parameters used. Table 4 below setsout the peak listings. TABLE 3 Manufacturer Philips Analytical X-Ray B.V. The Netherlands Diffractometer type PW3040/60 Serial Number DY1379Tube Anode Cu K-Alpha 1 Wavelength (Å) 1.54056 Divergence Slit Angle (°)0.1250 Receiving Slit None Monochromator used YES Generator Voltage 40Tube Current 45 Data Angle Range (°2θ) 5.000-45.000 Scan Step Size (°2θ)0.0128 Scan Type Continuous Scan Time per Step (s) 2999.33 SamplePreparation Sample filled into a capillary tube Detector Raytech PSD PSDmode Scanning PSD Number of active PSD 591.00 channels PSD pitch 0.0533Divergence slit Slit Fixed 1/8° Hybrid Monochromator 2xGe220 Asym.(hybrid) Mirror X-ray mirror Cu (hybrid MRD)

[0104] TABLE 4 Angle (°2θ) Relative intensity (%) 6.558 45 7.261 847.748 15 11.469 1 13.127 13 13.530 30 15.529 5 17.241 27 18.082 1718.288 12 19.688 6 19.867 7 20.122 77 20.302 40 20.884 34 21.877 6522.025 100 22.520 9 22.943 18 23.041 30 23.182 43 23.380 41 23.796 2424.422 17 24.607 7 24.934 9 25.569 23 26.156 35 26.432 7 27.347 7327.499 23 27.796 3 28.195 20 28.743 12 28.961 18 29.313 7 29.861 630.011 8 30.225 25 30.810 7 31.095 16 31.366 33 31.541 8 32.228 6 32.5857 33.260 13 33.473 13 34.113 5 34.387 1 34.676 7 34.866 12 35.020 735.284 3 36.287 6 36.578 8 36.761 17 37.054 4 37.530 13 37.733 5 38.3867 38.597 13 38.834 15 39.462 9 39.827 9 39.897 8 40.082 4 40.367 440.492 4 40.875 2 41.464 7 41.839 1 42.466 5 42.557 6 43.166 2 43.478 243.750 3 44.016 4 44.473 3 44.672 2

[0105] The hydroscopicity of a sample of the compound of formula (I)trihydrate prepared in accordance with the above method was tested bymeasuring % weight change (w/w) over a target relative humidity band of0 to 90% at 25° C. The instruments and parameters used were as forgeneration of the data shown in FIGS. 1 to 3. The results are shown inFIG. 5.

Comparative Example A

[0106] A dihydrochloride salt of the compound of which the compound offormula (I) is the phosphate salt was prepared by reacting anintermediate product of example 1, namely tert-butylN-(tert-butoxycarbonyl)-S-[2-(ethanimidoylamino)ethyl]-L-homocysteinatehydrochloride, with hydrogen chloride and dioxan at room temperature, asshown below.

[0107] The hygroscopicity of a sample of the dihydrochloride saltprepared in accordance with the above method was tested by measuring its% weight change (w/w) over a target relative humidity band of 30 to 80%at 25 degrees C. The results are shown in FIG. 4.

[0108] It can be seen that there was a rapid uptake of atmosphericmoisture above 50% relative humidity. At 55% relative humidity, thedihydrochloride salt had a mass change of about 11%; at 60% relativehumidity, the dihydrochloride salt had a mass change of about 24%; andat 65% relative humidity, the dihydrochloride salt had a mass change ofabout 32%. The mass change over a relative humidity range of 30-75% wasabout 47%.

1. A compound of formula (I) being

or a solvate or physiologically functional derivative thereof. 2.(2S)-2-amino-4-{[2-(ethanimidoylamino)ethyl]thio}butanoic acid, (1:1)compound with phosphoric acid, or a solvate or physiologicallyfunctional derivative thereof.
 3. A compound as claimed in claim 1 orclaim 2 wherein the phosphoric acid is orthophosphoric acid.
 4. Acompound as claimed in any of claims 1 to 3 wherein the solvate is ahydrate.
 5. A compound as claimed in claim 4 wherein the hydrate is themonohydrate.
 6. A compound as claimed in claim 4 wherein the hydrate isthe trihydrate.
 7. A method for the prophylaxis or treatment of aclinical condition in a mammal, such as a human, for which an inhibitorof nitric oxide synthase is indicated, which comprises administration ofa therapeutically effective amount of a compound as claimed in any ofclaims 1 to
 6. 8. A method according to claim 7 wherein the clinicalcondition is selected from arthritis, asthma, rhinitis, ileus, migraine,pain and irritable bowel syndrome.
 9. A compound as claimed in any ofclaims 1 to 6 for use in medical therapy.
 10. A pharmaceuticalformulation comprising a compound as claimed in any of claims 1 to 6 anda pharmaceutically acceptable carrier or excipient, and optionally oneor more other therapeutic ingredients.
 11. Use of a compound as claimedin any of claims 1 to 6 in the manufacture of a medicament for theprophylaxis or treatment of a clinical condition for which an inhibitorof nitric oxide synthase is indicated.
 12. Use according to claim 11wherein the clinical condition is selected from arthritis, asthma,rhinitis, ileus, migraine, pain and irritable bowel syndrome.
 13. Aprocess for preparing a compound as defined in claim 1 or claim 2 or asolvate, or physiologically functional derivative thereof whichcomprises the following steps: (i) reaction of the compound of formula(II)

or an enantiomer, a salt, or a protected derivative thereof, with acompound of formula (III)

or a salt thereof, wherein L is a leaving group; followed by thefollowing steps in any order: (ii) converting the resulting compound toa monophosphate salt; (iii) optional removal of any protecting groups;(iv) optional separation of an enantiomer from a mixture of enantiomers;(v) optional conversion of the product to a corresponding solvate orphysiologically functional derivative thereof.
 14. A process as claimedin claim 13, wherein the step of converting the resulting compound to amonophosphate salt comprises a biphasic reaction using toluene andaqueous phosphoric acid.
 15. A process as claimed in claim 13 or claim14, wherein, following step (i): toluene is added to an aqueous layerresulting from the reaction of a compound of formula (II) or anenantiomer, a salt, or a protected derivative thereof with a compound offormula (III) or a salt thereof to form a biphasic mixture; a toluenelayer is separated from this mixture; an aqueous solution of phosphoricacid is added to the toluene layer to form a further biphasic mixture;and an aqueous layer is separated from this further mixture.